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Study of MMLV RT-Binding with DNA using surface Plasmon Resonance Biosensor

Acta Biochimica et Biophysica Sinica 2005年第9期37卷 634-642页

作者： lei wu~1 ming-hui huang~2.jian-long zhao~(1*) meng-su yang~~(1,2*) 1 shanghai institute of microsystem and information technology,chinese academy of sciences,shanghai 2.0050,china 2.department of bioloy and chemistry,City university of hong kong,hong kong,china shanghaiinstituteof{2.andinformationtechnology ChineseAcademyofSciencesShanghai200050China

surface plasmon resonance biosensor technique was used to study the binding of Moloneymurine leukemia virus reverse transcriptase without RNase H domain （MMLV RT-） with DNA in the ab-sence and in the presence of *** DNA substrates,including single-stranded DNA （ssDNA）,DNA template-primer （T-P） duplex and gapped DNA,were immobilized on the biosensor chip surface usingstreptavidin-biotin,and MMLV RT--DNA binding kinetics were analyzed by different *** RT-could bind with ssDNA and the binding was involved in conformation *** RT- binding DNA T-Pduplex and gapped DNA could be analyzed using the simple 1:1 Langmuir *** lack of RNase Hdomain reduced the affinity between MMLV RT- and T-P *** effects of RT inhibitors,includingefavirenz,nevirapine and quercetin,on the interaction between MMLV RT- and gapped DNA were analyzedaccording to recovered kinetics *** slightly interfered with the binding between RT andDNA and the affinity constant in the presence of the inhibitor (KA=1.2.×106 M-1) was lower than in theabsence of the inhibitor (KA=4.61×106 M-1).Nevirapine induced relatively tight binding between RT andDNA and the affinity constant in the presence of the inhibitor (KA=1.47×107 M-1) was approximately threefolds higher than without nevirapine,mainly due to rapid association and slow ***,aflavonoid originating from plant which has previously shown strong inhibition of the activity of *** to have minimal effect on the RT-DNA binding.

关键词： surface plasmon resonance biosensor reverse transcriptase kinetics inhibitor

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